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Background and Aim: Obese state is associated with increased breast cancer growth, metastasis, and poor overall survival. In this study, we seek to decipher the underlying molecular mechanisms by which obesity/hyperleptinemia reduces the efficacy of tamoxifen. Methods: The impact of obesity on tamoxifen was evaluated utilizing clonogenicity, high-fat-diet- induced obese mice and leptin-treated xenograft-models. Mechanistic studies involved immunoblotting, real-time PCR, immunocytochemistry, chromatin immunoprecipitation assay, phosphokinase array and in silico analysis. Results: Obese mice with hyperleptinemia exhibit increased tumor progression and respond poorly to tamoxifen compared to non-obese mice. Exogenous leptin abrogates tamoxifen-mediated growth inhibition and potentiates breast tumor growth even in the presence of tamoxifen. Mechanistically, leptin induces nuclear translocation of phosphorylated-ER and increases the expression of ER-responsive genes while reducing tamoxifen-mediated gene repression by abrogating tamoxifen-induced recruitment of corepressors NCoR, SMRT and Mi2. Further, we found that coactivator Med1 potentially associates with 48 (out of 75) obesity-signature genes. Interestingly, leptin upregulates Med1 expression by decreasing miR-205 and increases its functional activation via phosphorylation that is mediated by activation of Her2 and EGFR. It is important to note that Med1 silencing abrogates the negative effects of leptin on tamoxifen efficacy. Additionally, honokiol or adiponectin treatment effectively inhibits leptin-induced Med1 expression and improve tamoxifen efficacy in hyperleptinemic state.Conclusion: In conclusion, these studies show the molecular mechanisms by which obese/hyperleptinemic state may contribute to poor response to tamoxifen implicating leptin-miR205-Med1 and leptin-Her2-EGFR-Med1 axes and present bioactive compound honokiol and adipocytokine adiponectin as agents that can block leptin’s negative effect on tamoxifen. Citation Format: Arumugam Nagalingam, Nethaji Muniraj, Sumit Siddharth, Dimiter Avtanski, Sheetal Parida, Pajamurthy Kuppusamy, Neeraj K Saxena, Nethaji Muniraj. Hyperleptinemia in obese state renders luminal breast cancers refractory to tamoxifen coordinating a crosstalk between Med1, miR205 and Erb B kinases [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-32.

Background and Aim: Obese state is associated with increased breast cancer growth, metastatic progression, poor overall survival and poor response to therapy. Analyses of clinical trial population showed that obesity associates with poor outcomes in patients with estrogen receptor (ER)-positive breast cancer treated with standard chemohormonal therapy. A characteristic feature of obesity is hyperleptinemia (high leptin levels) which mediates the biological effects of obesity. In this study, we seek to decipher the underlying molecular mechanisms by which obesity/hyperleptinemia reduces the efficacy of tamoxifen. Results: Obese mice models exhibiting hyperleptinemia supported larger tumors and responded poorly to tamoxifen therapy. Kaplan meier analyses showed that high leptin and high leptin receptor levels associates with poor prognosis in ER positive breast cancer. Indeed, leptin treatment abrogated tamoxifen-mediated inhibition of clonogenicity, and soft-agar colony formation. Leptin treatment increased breast tumor growth and significantly reduced the efficacy of tamoxifen. At the molecular level, leptin induced nuclear translocation of pER (ser 118 and ser 167) and increased the expression of ER-responsive genes while reducing tamoxifen-mediated gene repression. Tamoxifen is known to recruit corepressors to ER-responsive genes. ChIP analysis revealed that tamoxifen-induced recruitment of NCoR, SMRT and Mi2 corepressors was abrogated in the presence of leptin. We found that Med1, anchor subunit of human mediator complex, interacts with various key effector molecules of leptin-signaling network and potentially associates with 48 (out of 75) obesity-signature genes indicating its functional importance. Further, we found that leptin upregulated Med1 via decreasing miR-205 and increased its functional activation via phosphorylation, nuclear translocation and promoter-recruitment. Med1 phosphorylation was mediated by activation of Her2 and EGFR in response to leptin. It is important to note that silencing of Med1 abrogated the negative effects of leptin on tamoxifen efficacy. Bioactive strategies using honokiol or adiponectin treatment effectively inhibited leptin-induced Med1 expression and increased the efficacy of tamoxifen even in the presence of leptin. Conclusion: In conclusion, these studies show the molecular mechanisms by which obese state may contribute to poor response to endocrine therapy. We show that activation of Med1 via leptin-induced EGFR and Her2 kinases act as the key node mediating the negative effects of leptin on tamoxifen efficacy. A combination regimen of honokiol and adiponectin with tamoxifen may increase its efficacy in obese state. Citation Format: Arumugam Nagalingam, Nethaji Muniraj, Sumit Siddharth, Dimiter. B Avtanski, Sheetal Parida, Panjamurthy Kuppusamy, Balázs Győrffy, Neeraj Saxena, Dipali Sharma. Hyperleptinemia in obese state renders luminal breast cancers refractory to tamoxifen coordinating a crosstalk between Med1, miR205 and Erb B kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 310.

Background and Aim: Honokiol, a natural phenolic compound isolated from an extract of seed cones from Magnolia grandiflora, is widely known for its therapeutic potential as an antioxidant, anti-inflammatory, antithrombosis, and anti-depressant agent. Our recent studies show that honokiol impedes breast carcinogenesis. Cancer cells undergo cytoprotective autophagy and evade chemotherapy therefore many clinical trials are investigating the efficacy of autophagy inhibition in combination with chemotherapy. In the present study, we investigated the involvement of autophagic process in honokiol-mediated functional networks in breast cancer and explored the efficacy of combination regimens involving honokiol and chloroquine. Methods: Autophagy studies were conducted utilizing immunoblot, RT-PCR, immunofluorescence analyses, confocal imaging and transmission electron microscopy for autophagy markers. Autophagic flux was analyzed using a plasmid tfLC3B and acridine orange staining. The fusion of autophagosome and lysosome was examined by using GFP-LC3/LysoTracker-red. Functional impact of autophagic process was evaluated using genetic knockout (KO) of AGT7 and BECN1 in MCF7 cells as well as combined treatment with autophagy inhibitors (3-MA, Baf-1 and CQ) and honokiol. Alterations in ATP levels were measured by ATPliteTM luminescence assay. In vivo studies using mammary gland implantation of cancer cells in NOD-SCID mice were conducted to evaluate the efficacy of combination regimen of Honokiol and Chloroquine. Results: We found that Honokiol induces autophagy flux, increases accumulation of autophagosomes and elevates LC3B-II-conversion. Utilizing tandem-mCherry-GFP-LC3B assay and LysoTracker Red-staining, we observed that honokiol increases the autophagosome/lysosome fusion in breast cancer cells. We found that Honokiol induces autophagic response in a STK11-dependent manner as STK11-null breast cancer cells do not exhibit LC3B-II-puncta in response to Honokiol. Next, we explored the functional impact of autophagy in Honokiol mediated breast cancer inhibition and found that inhibiting autophagosome formation, abrogating autophagosome-lysosome fusion or genetic-knockout of BECN1/Beclin1 and ATG7 effectively increases the efficacy of Honokiol. These results clearly showed the cytoprotective nature of Honokiol-mediated autophagy and put forth the notion that a combined strategy of autophagy-inhibition with Honokiol would be more effective. Indeed, our in vivostudies showed that a combined treatment with Honokiol and Chloroquine can effectively inhibit primary tumor growth as well as metastatic progression. Conclusion: Together, these results implicate that honokiol is a potent inducer of cytoprotective autophagy and a combined treatment of honokiol and chloroquine is a promising therapeutic strategy for breast cancer. Citation Format: Nethaji Muniraj, Marey Shriver, Arumugam Nagalingam, Sumit Siddharth, Sheetal Parida, Neeraj K Saxena, Dipali Sharma. Induction of STK11-dependent cytoprotective autophagy in breast cancer cells upon Honokiol treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4271.

Introduction: Despite significant progress towards screening efforts and targeted therapies, breast cancer is still a leading cause of cancer-related mortality among women. Novel therapeutic strategies are clearly needed for effective targeting of breast cancer. Natural products continue to generate interest for identification of potential chemopreventive and therapeutic agents. Withaferin A (WFA) is a bioactive small molecule that has been proposed as a new-generation molecule capable of eliciting growth inhibitory effect on cancer cells. The molecular mechanisms by which WFA mediates inhibition of breast cancer remain elusive. The present study was designed to systematically elucidate the underlying mechanisms by which WFA inhibits growth and metastatic potential of breast cancer cells. Methods: Efficacy of WFA treatment on cell growth was evaluated by using clonogenicity, and anchorage–independent growth. Western blot and immunofluorescence analysis were used to examine activation of ERK/RSK (extracellular signal-regulated kinase/ ribosomal S6 kinase) and downstream signaling axes. Functional importance of ERK/RSK and CHOP (C/EBP homologous protein) in the biologic effects of WFA was examined by using overexpression, phospho-deficient constructs and specific inhibitors. Finally, mouse xenografts, immunohistochemical, RT-PCR and western blot analysis of tumors were used. Results: Analysis of the underlying molecular mechanisms revealed that WFA treatment induced phosphorylation of RSK2 in an ERK-dependent manner. Further, WFA-induced ERK/RSK signaling resulted in concomitant upregulation of CHOP and Elk-1(ETS-like transcription factor 1). By using ERK1/2 siRNA and FMK-MEA inhibitor, we found that ERK/RSK signaling axis is required for WFA-mediated modulation of CHOP and Elk-1. Intriguingly, we discovered that WFA triggers the nuclear translocation of CHOP and Elk1 resulting in transcriptional upregulation of Death Receptor protein (DR5). CHOP and Elk1 overexpression potentiated while phospho-deficient Elk-1 inhibited WFA-induced DR5 expression exhibiting that CHOP/Elk-1 cooperatively regulate DR5 expression. Furthermore, using nontoxic doses of WFA, we showed that WFA treatment effectively inhibited breast tumorigenesis in vivo. Analysis of WFA-treated breast tumors showed increased activation of ERK/RSK axis and DR5 along with higher nuclear accumulation of CHOP and Elk-1. Conclusion: These data provided first in vitro and in vivo evidence of the integral role of a previously unrecognized crosstalk between WFA and ERK/RSK and CHOP/Elk-1 axes in breast tumor growth inhibition. WFA treatment could potentially be a rational therapeutic strategy for breast carcinoma. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-12.

Introduction: Molecular effects of obesity, a well-established risk factor for breast cancer progression, are mediated by adipocytokine leptin. Recent studies from our lab reveal that leptin induces epithelial-mesenchymal transition (EMT) and tumorsphere formation via concomitant activation of Akt/GSK3b and MTA1/Wnt1 signaling axes leading to β catenin activation. Given the important role of leptin in breast cancer growth and metastasis, novel strategies to antagonize biological effects of leptin are much desired. We showed previously that honokiol, a bioactive polyphenol from Magnolia grandiflora, inhibits breast carcinogenesis. The present study provides first evidence for the efficacy of honokiol against oncogenic effects of leptin including EMT. Methods: Efficacy of honokiol to inhibit oncogenic effects of leptin was evaluated by using clonogenicity, anchorage–independent growth, matrigel-invasion and spheroid-migration assays. RT-PCR, western blot and immunofluorescence analyses were used to examine the molecular changes associated with EMT as well as underlying molecular pathways. Functional importance of MTA1/Wnt1/ β catenin axis was examined by using overexpression, phospho-deficient constructs and specific inhibitors. Finally, mouse xenografts, immuniohistochemical, RT-PCR and western blot analysis of tumors was used. Results: Honokiol treatment circumvents leptin-induced EMT-associated phenotypic changes. A biochemical hallmark of EMT is loss of expression of epithelial markers with a concurrent increase in mesenchymal marker expression. Honokiol elicits increased expression of E-cadherin, occludin and keratin-18 (epithelial markers) and decreased expression of vimentin, fibronectin and N-cadherin (mesenchymal markers) in leptin-treated cells providing molecular evidence for reversal of EMT. Honokiol also inhibits expression and nuclear translocation of transcriptional effectors of EMT; Snail, Slug, Zeb1 and Zeb2. Analysis of underlying molecular mechanisms reveals that Honokiol effectively inhibits leptin-induced MTA1/Wnt1/ β catenin axis. Furthermore, using nontoxic doses of honokiol, we show that honokiol treatment effectively inhibits leptin-induced breast tumorigenesis in vivo. Analysis of breast tumors show that honokiol treatment reverses leptin-associated signaling (MTA1/Wnt1/ β catenin axis) along with decreased expression of mesenchymal markers and increased expression of epithelial markers. Conclusions: In this study, we demonstrate for the first time that HNK is able to abolish leptin-induced EMT and provide in vitro and in vivo evidence for the integral role of a previously unrecognized crosstalk between honokiol and MTA1/Wnt1/ β catenin axis. Thus, HNK may be used as a non-toxic and non-endocrine rational therapeutic strategy for breast carcinoma in obese patients with high leptin levels. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-07-04.

Background and Aim: Cancer cells undergo cytoprotective autophagy and evade chemotherapy therefore many clinical trials are investigating the efficacy of autophagy inhibition in combination with chemotherapy. At the functional level, autophagic process can be cytoprotective, cytotoxic, cytostatic or nonprotective. We investigated strategies to convert cellular autophagic response to non-protective autophagy which does not interfere with therapeutic regimens exploiting bioactive molecules. Methods: Utilizing in vivo xenograft models, we established that Withaferin A (WA), a bioactive molecule from Withania Somnifera inhibits breast tumorigenesis. Autophagy studies were conducted utilizing immunoblot, RT-PCR, and immunofluorescence analyses for autophagy markers, transmission electron microscopy and confocal imaging. The fusion of autophagosome and lysosome was examined by using GFP-LC3/LysoTracker-red and GFP-LC3/mCherryRAB7A. Protein degradation activity of lysosomes and ATP levels were examined by DQ-BSA assay, Cathepsin activity and quantitative ATP assay. Results: WA inhibited growth and induced apoptosis in breast cancer cells resulting in inhibition of breast carcinogenesis in vivo. Although WA increased tumor suppressor LKB1 which is known to be involved in autophagy, WA-mediated increased cleavage of Light Chain 3 type II (LC3-II) and punctated LC3-II staining was LKB1-independent. The redistribution of EGFP-LC3 from cytosol to autophagosome indicated increased formation of autophagosomes in WA-treated cells. However, WA-induced increased autophagosome-formation was not mediated by increased activation of autophagy by upstream processes but was due to blockade of lysosomal-degradation as evident by higher level of sequestosome 1 (SQSTM1/p62) and decreased turnover of LC3. WA was found to be a potent lysosomal deacidification agent capable of blocking autophagic flux. Accordingly, inhibiting autophagy by blocking formation of autophagosomes or elevating lysosomal pH did not interfere with WA-mediated growth-inhibition. WA blocked autophagic flux decreasing recycling of cellular fuels leading to reduced energy supply. Investigating this alternative mechanism, we discovered that indeed, WA reduced ATP levels and increased phosphorylation of AMP-activated protein kinase (AMPK). Modulating substrates for tricarboxylic acid (TCA) cycle with methyl pyruvate protected WA-treated cells while 2DG potentiated WA-induced cell death. Conclusion: Our results indicate that WA induces a non-protective autophagy and blocks energy fuels in cancer cells by reducing ATP levels and inhibiting lysosomal acidification hence offering a three-pronged approach to facilitate cancer cell death. WA might be a useful strategic addition to chemotherapy regimens to evade cytoprotective effects of autophagy. Citation Format: Nethaji Muniraj, Arumugam Nagalingam, Neeraj K. Saxena, Dipali Sharma. Withaferin A induces nonprotective autophagy in a STK11-independent manner and mediates breast cancer inhibition via energetic impairment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1335.

Background and Aim: Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world and the third most common cause of cancer-related deaths. While multiple chemotherapeutic and targeted agents have been developed for other cancers, Sorafenib, an oral multikinase inhibitor, is the only approved agent for the treatment of advanced HCC. Clearly, it is imperative to develop novel effective therapeutic strategies for HCC to improve long-term survival of HCC patients. We found Withaferin A (WFA), a bioactive alkaloid derived from Withania somnifera, as an effective agent for HCC inhibition. Interestingly, we observed that WFA also induces autophagy in HCC cells. Autophagy can be cancer inducing or cancer inhibiting at the functional level. We aim to systemically investigate the anti-HCC efficacy of WFA, investigate the functional impact of autophagy and explore the efficacy of combined regimens of WFA and autophagic inhibitors. Methods: Using in vitro and in vivo models, we established that WFA inhibits liver tumorigenesis. tfLC3B, LC3B-GFP/Lysotracker and LC3B/Rab7 were used to examine the fusion of autophagosomes with lysosomes. Cathepsin-D activation and DQ-BSA assays were conducted to analyze the protein degradation activity in lysosomes. Multiple cell based assays and Chou-Talalay synergy quantification assays were conducted to analyze the synergistic effect of WFA with autophagic inhibitors. IHC staining was used to study autophagic markers in vivo. Results: WFA inhibited anchorage dependent cell growth in vitro and reduced the HCC tumor burden in vivo. In addition to inducing apoptotic cell death, WFA induced cleavage of Light Chain 3B (LC3B) and increased the expression of autophagic proteins (ATG5, ATG7 and Beclin1) both in vitro and in vivo. The redistribution of LC3B-GFP from cytosol to autophagosome demonstrated enhanced autophagosome formation in WFA treated cells. The fusion of autophagosome with lysosome was observed in WFA-treated HCC cells indicating autolysosome formation. WFA treated HCC cells exhibited elevated lysosomal degradation activity. Inhibiting autophagy either by blocking autophagosome formation or elevating lysosomal pH (CQ and BAF) enhanced WFA-induced growth inhibition and apoptosis indicating the presence of cytoprotective autophagy. Combination index analysis of WFA and CQ showed synergism and increased efficacy as compared to either monotherapy. These in vitro findings were corroborated with in vivo experiments. Conclusion: Our results reveal that WFA inhibits hepatocellular carcinoma by apoptotic induction but the efficacy of WFA is somewhat abrogated by the concomitant induction of cytoprotective autophagy. Our preclinical findings present evidence to support WFA and chloroquine as an effective combination treatment regimen for HCC. Citation Format: Sumit Siddharth, Nethaji Muniraj, Neeraj K. Saxena, Dipali Sharma. Withaferin A induces cytoprotective autophagy in hepatocellular cancer cells and concomitant autophagic inhibition augments the efficacy of Withaferin A in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1334.

Dicer is a well-conserved and essential gene that is required for the production of micro RNAs that play key roles during development and cancer. Several studies have associated aberrant Dicer expression in humans with developmental defects (Dicer1 syndrome) and poor prognosis in various cancers. One important mechanism of controlling protein stability and function is through post-translational modification. Recently, Extracellular signal-Regulated Kinase (ERK) dependent phosphorylation of Dicer during oogenesis has been shown to be essential in C. elegans. The phosphorylation sites in Dicer (Ser1718 and Ser1842 in mice) are well conserved and the phosphorylation is necessary and sufficient for Dicer's translocalization from cytoplasm to nucleus. We are interested in understanding the phosphorylation-mediated control of Dicer function during development and cancer. To understand the role of phospho-Dicer in mammals, we have generated five new Dicer1 alleles including phospho-mimetic mouse models with both serines replaced with aspartic acids (DicerS2D), only Ser1842 replaced with aspartic acid (DicerS1842D), and only Ser1718 replaced with aspartic acid (DicerS1718D). Phenotypic study shows that >75% of DicerS2D/S2D mutants and >60% of DicerS1842D/S1842D mutants die within five days after birth, while all heterozygous mutants and DicerS1718D/S1718D mice are phenotypically normal. The DicerS2D/S2D and DicerS1842D/S1842D survivors show signs of accelerated aging including smaller size, hunching of backbone, compromised weight gain, infertility, systemic tissue atrophy, and early lethality. We have also observed that DicerS2D/S2D animals and MEFs have increased metabolism as compared with wild type. Since aging and cancer are closely linked and altered metabolism is a hallmark of cancer, we hypothesize that phosphorylated Dicer produces an altered miRNA profile that affects metabolism, leading to developmental defects and tumorigenesis. Transcriptional profiling of MEFs and testes have shown that miRNA profiles are very different in mutants than in wild type, and the most affected pathways are metabolism and cancer. To test the role of phospho-Dicer in tumorigenesis, we crossed the DicerS2D allele into KRasLA1/+ driven nonmetastatic tumor model. The results show that phospho-Dicer reduces tumor-free survival, changes tumor spectrum, and drives metastasis in this tumor model. Citation Format: Neeraj K. Aryal, Swathi Arur, Guillermina Lozano. Phosphorylation results in nuclear translocation of Dicer protein that increases metabolism, accelerates aging, and alters tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A19.

Functional reactivation of p53 pathway, although arduous, can potentially provide a broad-based strategy for cancer therapy owing to frequent p53 inactivation in human cancer. Using a phosphoprotein-screening array, we found that Benzyl Isothiocynate, (BITC) increases p53 phosphorylation in breast cancer cells and reveal an important role of ERK and PRAS40/MDM2 in BITC-mediated p53 activation. We show that BITC rescues and activates p53-signaling network and inhibits growth of p53-mutant cells. Mechanistically, BITC induces p73 expression in p53-mutant cells, disrupts the interaction of p73 and mutant-p53, thereby releasing p73 from sequestration and allowing it to be transcriptionally active. Furthermore, BITC-induced p53 and p73 axes converge on tumor-suppressor LKB1 which is transcriptionally upregulated by p53 and p73 in p53-wild-type and p53-mutant cells respectively; and in a feed-forward mechanism, LKB1 tethers with p53 and p73 to get recruited to p53-responsive promoters. Analyses of BITC-treated xenografts using LKB1-null cells corroborate in vitro mechanistic findings and establish LKB1 as the key node whereby BITC potentiates as well as rescues p53-pathway in p53-wild-type as well as p53-mutant cells. These data provide first in vitro and in vivo evidence of the integral role of previously unrecognized crosstalk between BITC, p53/LKB1 and p73/LKB1 axes in breast tumor growth-inhibition. Citation Format: Arumugam Nagalingam, Bei Xie, Panjamurthy Kuppusamy, Nethaji Muniraj, Peter Langford, Balázs Győrffy, Neeraj Saxena, Dipali Sharma. Benzyl isothiocyanate potentiates p53 signaling and antitumor effects against breast cancer through activation of p53-LKB1 and p73-LKB1 axes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2576. doi:10.1158/1538-7445.AM2017-2576

Background and Aim: Cancer cells undergo cytoprotective autophagy and evade chemotherapy therefore many clinical trials are investigating the efficacy of autophagy inhibition in combination with chemotherapy. At the functional level, autophagic process can be cytoprotective, cytotoxic, cytostatic or nonprotective. We investigated strategies to convert cellular autophagic response to non-protective autophagy which does not interfere with therapeutic regimens exploiting bioactive molecules. Methods: Utilizing in vivo xenograft models, we established that Withaferin A (WA), a bioactive molecule from Withania Somnifera inhibits breast tumorigenesis. Autophagy studies were conducted utilizing immunoblot, RT-PCR, and immunofluorescence analyses for autophagy markers, transmission electron microscopy and confocal imaging. The fusion of autophagosome and lysosome was examined by using GFP-LC3/LysoTracker-red and GFP-LC3/mCherryRAB7A. Protein degradation activity of lysosomes and ATP levels were examined by DQ-BSA assay, Cathepsin activity and quantitative ATP assay. Results: WA inhibited growth and induced apoptosis in breast cancer cells resulting in inhibition of breast carcinogenesis in vivo. Although WA increased tumor suppressor LKB1 which is known to be involved in autophagy, WA-mediated increased cleavage of Light Chain 3 type II (LC3-II) and punctated LC3-II staining was LKB1-independent. The redistribution of EGFP-LC3 from cytosol to autophagosome indicated increased formation of autophagosomes in WA-treated cells. However, WA-induced increased autophagosome-formation was not mediated by increased activation of autophagy by upstream processes but was due to blockade of lysosomal-degradation as evident by higher level of sequestosome 1 (SQSTM1/p62) and decreased turnover of LC3. WA was found to be a potent lysosomal deacidification agent capable of blocking autophagic flux. Accordingly, inhibiting autophagy by blocking formation of autophagosomes or elevating lysosomal pH did not interfere with WA-mediated growth-inhibition. WA blocked autophagic flux decreasing recycling of cellular fuels leading to reduced energy supply. Investigating this alternative mechanism, we discovered that indeed, WA reduced ATP levels and increased phosphorylation of AMP-activated protein kinase (AMPK). Modulating substrates for tricarboxylic acid (TCA) cycle with methyl pyruvate protected WA-treated cells while 2DG potentiated WA-induced cell death. Conclusion: Our results indicate that WA induces a non-protective autophagy and blocks energy fuels in cancer cells by reducing ATP levels and inhibiting lysosomal acidification hence offering a three-pronged approach to facilitate cancer cell death. WA might be a useful strategic addition to chemotherapy regimens to evade cytoprotective effects of autophagy. Citation Format: Nethaji Muniraj, Arumugam Nagalingam, Panjamurthy Kuppusamy, Neeraj K. Saxena, Dipali Sharma. A three-pronged attack on cancer cells: Induction of non-protective autophagy, inhibition of lysosomal acidification and promotion of energetic impairment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3301. doi:10.1158/1538-7445.AM2017-3301

Adiponectin, an adipocytokine secreted by adipocytes in the breast tumor microenvironment, negatively regulates cancer cell growth hence increased levels of adiponectin are associated with decreased breast cancer growth. However, its mechanisms of action remain largely elusive. We report that adiponectin induces a robust accumulation of autophagosomes, increases LC3II and decreases p62/SQSTM1 in breast cancer cells. Adiponectin-treated cells and xenografts exhibit increased expression of autophagy-related proteins. Lysotracker-Red-staining and tandem-mCherry-GFP-LC3 assay show that autophagosomes/lysosomes fusion is augmented upon adiponectin treatment. Adiponectin significantly inhibits breast cancer growth and induces apoptosis both in vitro and in vivo, and these events are preceded by autophagy, which is integral for adiponectin-mediated cell death. Accordingly, blunting autophagosomes-formation, blocking autophagosomes-autolysosome fusion or genetic-knockout of BECN1/Beclin1/ATG7 effectively impedes adiponectin induced growth-inhibition and apoptosis-induction. Mechanistic studies show that adiponectin reduces intracellular ATP levels and increases AMPK phosphorylation leading to ATG1 activation. AMPK-inhibition abrogates adiponectin-induced ATG1-activation, LC3II-turnover and p62-degradation while AMPK-activation potentiates adiponectin’s effects. Further, adiponectin-mediated AMPK-activation and autophagy-induction are regulated by upstream master-kinase LKB1, which is a key node in anti-tumor function of adiponectin as LKB1-knockout abrogates adiponectin-mediated inhibition of breast tumorigenesis and molecular analyses of tumors corroborate in vitro mechanistic findings. Adiponectin increases the efficacy of chemotherapeutic agents. Notably, high expression of adiponectin receptor, adiponectin and BECN1 significantly correlates with increased overall survival in chemotherapy-treated breast cancer patients. Collectively, these data uncover that adiponectin induces autophagic cell death in breast cancer and provide in vitroa and in vivo evidence for the integral role of LKB1-AMPK-ATG1 axis in adiponectin-mediated cytotoxic-autophagy. Citation Format: Seung J Chung, Ganji Purnachandra Nagaraju, Arumugam Nagalingam, Nethaji Muniraj, Panjamurthy Kuppusamy, Alyssa Walker, Juhyung woo, Balázs Győrffy, Edward Gabrielson, Neeraj K. Saxena, Dipali Sharma. Elevating adipokine adiponectin level can induce cytotoxic autophagy in breast cancer cells and potentiate the efficacy of chemotherapeutic regimens: preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3319. doi:10.1158/1538-7445.AM2017-3319

Withaferin A (WFA) is a steroidal lactone with antitumor effects manifested at multiple levels that are mechanistically obscure. Using a phospho-kinase screening array, we discovered that WFA activated phosphorylation of the S6 kinase RSK (ribosomal S6 kinase) in breast cancer cells. Pursuing this observation, we defined activation of extracellular signal–regulated kinase (ERK)–RSK and ETS-like transcription factor 1 (Elk1)–CHOP (C-EBP homologous protein) kinase pathways in upregulating transcription of the death receptor 5 (DR5). Through this route, WFA acted as an effective DR5 activator capable of potentiating the biologic effects of celecoxib, etoposide, and TRAIL. Accordingly, WFA treatment inhibited breast tumor formation in xenograft and mouse mammary tumor virus (MMTV)-neu mouse models in a manner associated with activation of the ERK/RSK axis, DR5 upregulation, and elevated nuclear accumulation of Elk1 and CHOP. Together, our results offer mechanistic insight into how WFA inhibits breast tumor growth. Cancer Res; 74(9); 2617–29. ©2014 AACR.

Breast tumors expressing estrogen receptor-α (ER) respond well to therapeutic strategies using selective ER modulators, such as tamoxifen. However, ∼30% of invasive breast cancers are hormone independent because they lack ER expression due to hypermethylation of ER promoter. Treatment of ER-negative breast cancer cells with demethylating agents [5-aza-2′-deoxycytidine (5-aza-dC)] and histone deacetylase (HDAC) inhibitors (trichostatin A) leads to expression of ER mRNA and functional protein. Here, we examined whether epigenetically reactivated ER is a target for tamoxifen therapy. Following treatment with trichostatin A and 5-aza-dC, the formerly unresponsive ER-negative MDA-MB-231 breast cancer cells became responsive to tamoxifen. Tamoxifen-mediated inhibition of cell growth in these cells is mediated at least in part by the tamoxifen-bound ER. Tamoxifen-bound reactivated ER induces transcriptional repression at estrogen-responsive genes by ordered recruitment of multiple distinct chromatin-modifying complexes. Using chromatin immunoprecipitation, we show recruitment of two different corepressor complexes to ER-responsive promoters in a mutually exclusive and sequential manner: the nuclear receptor corepressor-HDAC3 complex followed by nucleosome remodeling and histone deacetylation complex. The mechanistic insight provided by this study might help in designing therapeutic strategies directed toward epigenetic mechanisms in the prevention or treatment of breast cancer. (Cancer Res 2006; 66(12): 6370-8)

Introduction: Despite significant progress towards screening efforts and targeted therapies, breast cancer is still a leading cause of cancer-related mortality among women. Novel therapeutic strategies are clearly needed for effective targeting of breast cancer. Natural products continue to generate interest for identification of potential chemopreventive and therapeutic agents. Withaferin A (WFA) is a bioactive small molecule that has been proposed as a new-generation molecule capable of eliciting growth inhibitory effect on cancer cells. The molecular mechanisms by which WFA mediates inhibition of breast cancer remain elusive. The present study was designed to systematically elucidate the underlying mechanisms by which WFA inhibits growth and metastatic potential of breast cancer cells. Results: Because many cellular signaling events involve induced-phosphorylation of key targets, in the present study we utilized phosphokinase arrays to gain insight into the intricacies of WFA-mediated signaling networks and discovered that WFA induces RSK phosphorylation in breast cancer cells. Further analysis revealed an integral role of ERK in WFA-mediated RSK activation and WFA-induced ERK/RSK signaling resulted in concomitant upregulation and activation of Elk1(ETS-like transcription factor-1) and CHOP (C-EBP homology protein) leading to transcriptional upregulation of Death Receptor protein-5 (DR5). It is interesting to note that in recent years, many phase 1 and 2 single agent and combination clinical trials have been conducted to examine the efficacy and safety of recombinant human TRAIL (dulanermin, (Amgen/Genentech)), and the agonistic monoclonal antibodies to DR5 (lexatumumab (Human Genome Sciences), conatumumab (Amgen), drozitumab (Genentech), tigatuzumab (Daiichi-Sankyo) and LBY135 (Novartis)). Studies with these pro-apoptotic receptor agonists (PARAs) have been encouraging but no full phase 3 studies have been performed suggesting the need for novel PARAs with improved properties. Here, we show that WFA is more effective than three other known DR5 activators, TRAIL, etoposide and celecoxib, in activation of DR5 in breast cancer cells hence, appears to be a good candidate for further development as an effective DR5 inducer. WFA treatment effectively inhibited breast tumorigenesis in vivo and analysis of WFA-treated xenografts and MMTV-neu mice tumors showed increased activation of ERK/RSK axis and DR5 along with elevated nuclear accumulation of Elk1 and CHOP. Conclusion: These data provided first in vitro and in vivo evidence of the integral role of a previously unrecognized crosstalk between WFA and ERK/RSK and CHOP/Elk1 axes in breast tumor growth inhibition. Also, our findings may potentially open new avenues of research on the role of withaferin A as a novel pro-apoptotic receptor agonist (PARA). Citation Format: Arumugam Nagalingam, Panjamurthy Kuppusamy, Neeraj Saxena, Dipali Sharma. A novel role of Withaferin A as an effective pro-apoptotic receptor agonist (PARA) and insights into the underlying molecular mechanisms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-113. doi:10.1158/1538-7445.AM2014-LB-113

Introduction: Dedifferentiation involves terminally differentiated cells reverting back to less differentiated cells such as precursor cells or stem cells within their own lineage allowing the cells to proliferate again before re-differentiation. Tumorigenesis involves uncontrolled cell proliferation and many host-related factors aid in dedifferentiation of tumor cells into stem cell like phenotypes hence providing a mechanism leading to tumor heterogeneity and aggressiveness. Pluripotency factors Oct4, Nanog and Sox2 have been implicated in dedifferentiation and are highly expressed in tumors. The importance of active constitutive agents in natural products has become increasingly apparent owing to their potential cancer chemopreventive and therapeutic properties. Honokiol (HNK) is a natural phenolic compound isolated from an extract of seed cones from Magnolia grandiflora. Recent studies from our laboratory demonstrated that HNK has suppressing effects on different aspects of cancer progression. The present study was designed to specifically examine the potential of HNK to inhibit pluripotency related transcription factors and reverse the process of dedifferentiation associated with uncontrolled cell proliferation in tumorigenesis. Results: Here, we provide evidence that HNK inhibits the ability of breast cancer cells to form mammospheres. HNK treatment inhibits dedifferentiation/pluripotency markers Oct4, Nanog, Sox2 in MCF-7, MDA-MB-231, T47D, MDA-MB-468 breast cancer cells. An analysis of underlying signaling mechanism reveal that HNK inhibits phosphorylation of Stat3 and the downregulation of pluripotency factors is mediated via inhibition of Stat3. Stat3 inhibitor, Stattic potentiates the effect of HNK while overexpression of constitutively active Stat3 interferes with HNK-mediated inhibition of Oct4, Nanog and Sox2. It is interesting to note that HNK treatment also increases the expression of upstream kinase and tumor suppressor LKB1. Utilizing LKB1-null cells and gain-of-function strategies, we show that HNK-mediated inhibition of Oct4, Nanog and Sox2 is regulated by tumor suppressor LKB1. The ability of HNK to inhibit mammosphere formation is abrograted in breast cancer cells stably-silenced for LKB1. In vitro and in vivo analyses show novel functional interactions between HNK, Stat3, LKB1 and dedifferentiation/pluripotency markers. Conclusions: Taken together, these studies provide evidence for a previously unrecognized cross-talk between HNK and dedifferentiation/pluripotency markers Oct4, Nanog, Sox2 via tumor suppressor LKB1-Stat3 axis in inhibiting “dedifferentiation” in breast cancer. Citation Format: Sonali Sengupta, Michael Y. Bonner, Jack L. Arbiser, Neeraj K. Saxena, Dipali Sharma. Inhibiting “Dedifferentiation” in breast cancer cells using Honokiol - a plant derived polyphenol. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-61. doi:10.1158/1538-7445.AM2014-LB-61

Introduction: An emerging hypothesis is that epithelial-mesenchymal-transition (EMT) bestows metastatic potential to epithelial cancer cells enabling them to invade, migrate and subsequently disseminate to form distant metastases resulting in non-curable disease. Pharmacological inhibition of EMT can potentially lead to reversion of aggressive breast cancer cells to a more-differentiated epithelial phenotype by inducing mesenchymal-epithelial transition (MET) to prevent metastasis and diminish distant recurrence. The importance of active constitutive agents in natural products has become increasingly apparent owing to their potential cancer chemopreventive and therapeutic properties. Honokiol (HNK) is a natural phenolic compound isolated from an extract of seed cones from Magnolia grandiflora. Recent studies from our lab demonstrated that HNK has suppressing effects on different aspects of cancer progression. The present study was designed to specifically examine the potential of HNK to inhibit EMT and elucidate the molecular mechanism by which HNK inhibits EMT in breast cancer cells. Aim: To elucidate the mechanism by which HNK inhibits EMT in breast cancer cells. Results: Here, we provide molecular evidence that HNK inhibits EMT in breast cancer cells resulting in significant downregulation of mesenchymal marker proteins and concurrent upregulation of epithelial markers. In vitro and in vivo analyses show functional interactions between HNK, STAT3, and EMT-signaling components. Mechanistically, HNK inhibits recruitment of STAT3 on mesenchymal transcription factor ZEB1 promoter resulting in decreased ZEB1 expression and nuclear translocation. We also discover that HNK increases E-cadherin expression via STAT3-mediated release of ZEB1 from E-cadherin promoter. Since ZEB1 is a transcriptional repressor of miR-34a, we next examined the effect of HNK on miR-34a expression. Intriguingly, we found that HNK upregulates the levels of miR-34a in a time- and dose-dependent manner. It is interesting to note that HNK treatment also increases the expression of upstream kinase and tumor suppressor LKB1. Utilizing LKB1-null cells and gain-of-function strategies, we found that HNK-mediated increase in miR-34a is regulated by tumor suppressor LKB1. Conclusions: Taken together, these studies provide evidence for a previously unrecognized cross-talk between HNK and miR-34a via tumor suppressor LKB1 in inhibiting EMT in breast cancer. Citation Format: Dimiter B. Avtanski, Michael Y. Bonner, Timothy P. Tiutan, Jack L. Arbiser, Neeraj K. Saxena, Dipali Sharma. Novel mechanistic insights into the bioactive compound honokiol-mediated inhibition of epithelial to mesenchymal transition in breast cancer: Therapeutic modulation of miR-34a via tumor suppressor LKB1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-187. doi:10.1158/1538-7445.AM2014-LB-187

Introduction: The epithelial-mesenchymal transition (EMT) presents a critical step in the acquisition of metastatic state. Tumor cells undergoing EMT not only exhibit increased migration and invasion potential but also acquire increased resistance to chemotherapy and radiation therapy. Hence, EMT is an attractive target for therapeutic interventions directed against tumor metastasis. Honokiol (HNK) is a natural phenolic compound isolated from an extract of seed cones from Magnolia grandiflora. Recent studies from our lab show that honokiol inhibits breast carcinogenesis. The present study is designed to systematically elucidate if nontoxic lower doses of honokiol can be used to specifically inhibit EMT in breast cancer cells. Results: Our studies show that HNK inhibits EMT in human breast cancer cells. Exposure of estrogen-independent and estrogen-responsive breast cancer cells to nontoxic lower doses of HNK results in significant downregulation of mesenchymal marker proteins (Fibronectin, Vimentin) along with simultaneous upregulation of epithelial markers (E-cadherin, Cytokeratin-18). HNK also inhibits expression and nuclear translocation of transcriptional modulators of EMT-related genes (Snail, ZEB1/2 and Twist). Experimental EMT induced by exposure to TGFβ and TNFα in a spontaneously immortalized nontumorigenic human mammary epithelial cell line (MCF-10A) was also completely reversed by HNK treatment as evidenced by morphological changes (inhibition of fibroblast-like appearance, decreased pseudopodia and actin reorganization to membrane-bound location in TGFβ and TNFα treated MCF10A cells) as well as molecular changes (downregulation of Fibronectin, Vimentin, Snail, Zeb1/2 and upregulation of E-cadherin and Cytokeratin-18). Mechanistically, HNK inhibits MTA1-Wnt1 axis leading to stabilization and nuclear translocation of β-catenin to inhibit EMT. Analysis of breast tumors treated with honokiol show modulation of EMT markers and inhibition of key molecules of MTA1-Wnt1-β-catenin axis corroborating our in vitro findings. Conclusions: Taken together, these data provide the first in vitro and in vivo evidence of the potential of HNK as a novel, non-toxic and non-endocrine therapeutic strategy for breast carcinoma. MTA1, Wnt1 and β-catenin appear to be novel biomarkers associated with HNK treatment. Citation Format: Dimiter B. Avtanski, Arumugam Nagalingam, Panjamurthi Kupusamy, Neeraj K. Saxena, Dipali Sharma. Targeting epithelial-mesenchymal transition in breast cancer cells using Honokiol, a natural phenolic compound. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 299. doi:10.1158/1538-7445.AM2013-299

Introduction: Obesity is an independent risk factor for the development of breast cancer. Molecular effects of obesity are mediated via perturbations in the adipocytokine profile. Adiponectin is widely considered as an adipocytokine with therapeutic potential as it inhibits growth of breast cancer cells but the mechanisms underlying growth inhibitory effects of adiponectin are still elusive. The present study was designed to systematically elucidate the underlying mechanisms by which adiponectin inhibits growth of breast cancer cells. Methods: Characteristic of autophagy were evaluated using transmission electron microscopy, acridine orange staining and western blot analysis of key effector molecules of autophagy. RT-PCR, western blot and immunofluorescence analysis were used to examine SIRT1 (Sirtuin 1), liver kinase B1 (LKB1), and AMP-activated protein kinase (AMPK) axes. Functional importance of AMPK activation and LKB1 overexpression in the biologic effects of adiponectin was examined by using AMPK-null and AMPK-wild type (WT) immortalized mouse embryonic fibroblasts (MEFs) and isogenic LKB1-knockdown cell line pairs. LKB1 immunoprecipitates were used to examine LKB1 deacetylation in response to adiponectin. Results: We provide strong evidence that adiponectin causes autophagy in breast cancer cells. Adiponectin treated breast cancer cells exhibit autophagosomes and major autophagosomal protein level changes including ATG1 (autophagy related 1) activation, cleavage of LC3 and suppression of p62 (p62/SQSTM1) expression. Adiponectin-induced autophagy leads to breast cancer cell death as evident by increased levels of cleaved-caspase 9, cleaved-PARP, and Tunel positivity. Adiponectin-mediated autophagy-induction as well as autophagic cell death is attenuated in the presence of autophagy inhibitors. Analysis of the underlying molecular mechanisms reveals that adiponectin treatment increases AMPK activation that is required for adiponectin-mediated ATG1 overexpression and phosphorylation. Intriguingly, we discover that adiponectin increases SIRT1 expression which deacetylates LKB1 leading to its activation. LKB1 knock-down inhibits adiponectin mediated autophagy induction as evidenced by modulations in major autophagosomal protein levels including ATG1. Analysis of breast tumors treated with adiponectin reveals significant increase in the levels of SIRT1, LKB1, phospho AMPK, phospho ATG1 as well as decreased p62. Conclusions: These data uncover a novel role of adiponectin as an inducer of autophagic cell death and provide the first in vitro and in vivo evidence of the integral role of the SIRT1-LKB1-ATG1 axis in adiponectin mediated autophagic cell death of breast cancer cells. Citation Format: Seung J. Chung, Neeraj Saxena, Dipali Sharma. Adiponectin induces autophagic cell death in breast cancer cells through SIRT1 mediated deacetylation of LKB1 leading to ATG1 activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1673. doi:10.1158/1538-7445.AM2013-1673

Introduction: Molecular effects of obesity, a well-established risk factor for breast cancer progression, are mediated by adipocytokine leptin. Recent studies from our lab reveal that leptin induces epithelial-mesenchymal transition (EMT) and tumorsphere formation via concomitant activation of Akt/GSK3β and MTA1/Wnt1 signaling axes leading to β-catenin activation. Given the important role of leptin in breast cancer growth and metastasis, novel strategies to antagonize biological effects of leptin are much desired. We showed previously that honokiol (HNK), a bioactive polyphenol from Magnolia grandiflora, inhibits breast carcinogenesis. The present study provides first evidence for the efficacy of HNK against oncogenic effects of leptin including EMT. Methods: Efficacy of HNK to inhibit oncogenic effects of leptin was evaluated by using clonogenicity, anchorage-independent growth, matrigel invasion and spheroid-migration assays. RT-PCR, Western blot and immunofluorescence analyses were used to examine the molecular changes associated with EMT as well as underlying molecular pathways. Functional importance of MTA1/Wnt1/β-catenin axis was examined by using overexpression, phospho-deficient constructs and specific inhibitors. Finally, mouse xenografts, immuniohistochemical, RT-PCR and Western blot analysis of tumors was used. Results: HNK treatment circumvents leptin-induced EMT-associated phenotypic changes. A biochemical hallmark of EMT is loss of expression of epithelial markers with a concurrent increase in mesenchymal marker expression. HNK elicits increased expression of E-cadherin, occludin and cytokeratin-18 (epithelial markers) and decreased expression of vimentin, fibronectin and N-cadherin (mesenchymal markers) in leptin-treated cells providing molecular evidence for reversal of EMT. HNK also inhibits expression and nuclear translocation of transcriptional effectors of EMT: Snail, Slug, Zeb1 and Zeb2. Analysis of underlying molecular mechanisms reveals that HNK effectively inhibits leptin-induced MTA1/Wnt1/β-catenin axis. Furthermore, using nontoxic doses of HNK, we show that HNK treatment effectively inhibits leptin-induced breast tumorigenesis in vivo. Analysis of breast tumors show that HNK treatment reverses leptin-associated signaling (MTA1/Wnt1/β-catenin axis) along with decreased expression of mesenchymal markers and increased expression of epithelial markers. Conclusions: In this study, we demonstrate for the first time that HNK is able to abolish leptin-induced EMT and provide in vitro and in vivo evidence for the integral role of a previously unrecognized crosstalk between honokiol and MTA1/Wnt1/ β catenin axis. Thus, HNK may be used as a non-toxic and non-endocrine rational therapeutic strategy for breast carcinoma in obese patients with high leptin levels. Citation Format: Dimiter B. Avtanski, Arumugam Nagalingam, Panjamurthy Kuppusamy, Neeraj K. Saxena, Dipali Sharma. A novel bioactive approach to inhibit leptin-induced epithelial-mesenchymal transition in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5497. doi:10.1158/1538-7445.AM2013-5497

Introduction: Research over the past few years have established LKB1-AMPK pathway as an important metabolic checkpoint interconnecting the signaling pathways controlling metabolism and cancer cell growth. Thus, it is of major interest to find effective therapeutic strategies to manipulate LKB1-AMPK axis to inhibit cancer growth. The importance of active constitutive agents in natural products has become increasingly apparent owing to their potential cancer preventive as well as therapeutic properties. Honokiol, a small-molecule polyphenol isolated from magnolia species, is widely known for its therapeutic potential as an anti-inflammatory, anti-thrombosis, anti-oxidant agent and more recently for its protective function in carcinogenesis. In the present study, we sought to examine the effectiveness of honokiol in modulating LKB1-AMPK axis, preventing mammary tumorigenesis and inhibiting metastasis. Methods: Clonogenicity, 3D-colony formation, matrigel invasion, scratch-migration and spheroid-migration assays were used to examine breast cancer cell growth and metastatic potential upon honokiol treatment. Western blot and immunofluorescence analysis were used to examine activation of LKB1-AMPK axis. Functional importance of AMPK and LKB1 was examined by using AMPK-null and AMPK-WT immortalized mouse embryonic fibroblasts (MEFs) and isogenic LKB1-knockdown cell line pairs. Breast cancer xenografts, immunohistochemical and western blot analysis of tumors were used. Results: Analysis of the underlying molecular mechanisms revealed that honokiol treatment increases AMP-activated protein kinase (AMPK) phosphorylation and activity as evident by increased phosphorylation of downstream target of AMPK, acetyl-coenzyme A carboxylase (ACC) and inhibition of phosphorylation of p70S6kinase (pS6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1). Using AMPK-null and AMPK-WT (MEFs); we found that AMPK is required for honokiol-mediated modulation of pACC-pS6K. Intriguingly, we discovered that honokiol treatment increased the expression and cytoplasmic translocation of tumor suppressor gene LKB1 in breast cancer cells. LKB1 knockdown inhibited honokiol-mediated activation of AMPK and more importantly, inhibition of migration and invasion of breast cancer cells. Furthermore, honokiol treatment resulted in inhibition of breast tumorigenesis in vivo. Analysis of tumors showed significant increase in the levels of cytoplasmic LKB1 and phospho-AMPK in honokiol-treated tumors. Conclusions: Taken together, these data providing first in vitro and in vivo evidence of the efficacy of honokiol in activating LKB1-AMPK axis resulting in effective inhibition of mammary tumorigenesis and metastasis provide impetus to determine its activity in clinical setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 153. doi:1538-7445.AM2012-153

Perturbations in the adipocytokines-profile, especially higher levels of leptin, are a major cause of breast tumor progression and metastasis; the underlying mechanisms, however, are not well understood. In particular, it remains elusive whether leptin is involved in epithelial-mesenchymal transition (EMT). Here, we provide first molecular evidence that leptin induces breast cancer cells to undergo a transition from epithelial to spindle-like mesenchymal morphology. Investigating the downstream mediator(s) that may direct leptin-induced EMT, we found functional interactions between leptin, metastasis-associated protein 1 (MTA1) and Wnt1 signaling components. Leptin increases accumulation and nuclear translocation of β-catenin leading to increased promoter recruitment. Silencing of β-catenin or treatment with small molecule inhibitor, ICG-001, inhibits leptin-induced EMT, invasion and tumorsphere formation. Mechanistically, leptin stimulates phosphorylation of glycogen synthase kinase 3β (GSK3β) via Akt activation resulting in a substantial decrease in the formation of GSK3β-LKB1-Axin complex which leads to increased accumulation of β-catenin. Leptin treatment also increases Wnt1 expression that contributes to GSK3β phosphorylation. Inhibition of Wnt1 abrogates leptin-stimulated GSK3β phosphorylation. We also discovered that leptin increases the expression of an important modifier of Wnt1 signaling, MTA1, which is integral to leptin-mediated regulation of Wnt/β-catenin pathway as silencing of MTA1 inhibits leptin-induced Wnt1 expression, GSK3β phosphorylation and β-catenin activation. Furthermore, analysis of leptin-treated breast tumors show increased expression of Wnt1, pGSK3β and Vimentin along with higher nuclear accumulation of β-catenin and reduced E-cadherin expression providing in vivo evidence for a previously unrecognized crosstalk between leptin and MTA1/Wnt signaling in epithelial-mesenchymal transition of breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 349. doi:1538-7445.AM2012-349

Obesity is a well-established risk factor for breast cancer progression and molecular effects of obesity are mediated by adipocytokine, leptin. Considering the important role of leptin in breast cancer growth and metastasis, novel strategies to antagonize biological effects of this adipocytokine are much desired. We showed previously that benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables (e.g., gardencress), confers significant protection against mammary carcinogenesis in a transgenic mouse model (MMTV-neu). The BITC-mediated prevention of mammary cancer development in MMTV-neu mice correlated with reduced cell proliferation, increased apoptosis, and tumor infiltration of T cells. The present study provides first evidence for efficacy of BITC against oncogenic effects of letpin. BITC-treatment circumvented leptin-induced clonogenicity and anchorage-independent growth of MDA-MB-231 (an estrogen-independent cell line with mutant p53) and MCF-7 human breast cancer cells (an estrogen-responsive cell line with wild-type p53) at pharmacologic doses. Leptin-stimulated migration and invasion of these cells was also inhibited significantly in the presence of BITC. Analysis of the underlying molecular mechanisms revealed that BITC-treatment suppressed leptin-induced phosphorylation of signal transducer and activator of transcription 3 (Stat3) and cyclin D1 transactivation. BITC-treatment efficiently inhibited Stat3 recruitment to the cyclin D1 promoter in a chromatin immunoprecipitation analysis. Furthermore overexpression of constitutively active Stat3 imparted significant protection against BITC-mediated inhibition of cyclin D1 transactivation whereas RNA interference of Stat3 resulted in a significant increase in BITC-mediated inhibition of cyclin D1 transactivation in the presence of leptin. BITC-mediated inhibition of MDA-MB-231 xenograft growth correlated with a decrease in level of phospho-Stat3. These studies indicate that Stat3 plays an important role in BITC-mediated inhibition of leptin-induced cyclin D1 transactivastion. Taken together, these data show that BITC treatment can inhibit the oncogenic actions of leptin via blocking its downstream signaling molecule, Stat3. In conclusion, BITC could potentially be a rational therapeutic strategy for breast carcinoma in obese patients with high leptin levels. This work was supported in part by the USPHS grants NCI-RO1 CA129347 (to SVS), NIDDK-K01DK076742 and R03DK089130 (to NKS), NCI-R01CA131294 and NCI-R21 CA155686 (to DS). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 860. doi:10.1158/1538-7445.AM2011-860

Background: Gene expression studies have identified two biologically distinct estrogen receptor (ER)-positive subtypes of breast cancer: luminal A and luminal B. Some luminal B tumors can be identified by their expression of Her2 but the major biological distinction between luminal A and luminal B is their proliferation signature. Upon treatment with adjuvant tamoxifen, luminal A breast tumors show a good prognosis, however the clinical outcome of luminal B breast tumor is poor. The underlying mechanisms for the differential response to adjuvant tamoxifen therapy between luminal A and luminal B breast tumors are probably multifactorial and remain largely unknown. In this study, we examined the molecular mechanisms contributing to the poor prognosis of the luminal B ER-positive breast cancer subtype in response to tamoxifen. Methods and Results: We developed a physiologically relevant luminal B ER-positive breast cancer (luminal B) model. This cell line model exhibited an elevated growth factor signaling pathway. We showed that tamoxifen treatment inhibited clonogenicity and anchorage-independent growth of luminal A breast cancer cells. Interestingly, acting as a potent agonist, tamoxifen resulted in significantly increased clonogenicity and anchorage-independent growth in luminal B cells. We found that tamoxifen also increased expression of ER-responsive genes in luminal B cells in contrast to luminal A cells where it effectively inhibited ER-transactivation and ER-responsive gene expression. Mediator complex is an essential coactivator complex for the expression of ER-responsive genes. Investigating the underlying mechanisms, we discovered that anchor subunit of mediator coactivator complex, Med1, was overexpressed in luminal B cells. Furthermore, we found hyper-phosphorylation of Med1 and ER in luminal B cells due to persistent activation of ERK. Hyper-phosphorylated Med1 showed increased recruitment to ER-responsive promoters in the presence of tamoxifen in luminal B cells. Stably silencing Med1 in luminal B cells not only inhibited tamoxifen-induced clonogenicity and anchorage-independent growth, it also inhibited tamoxifen-induced ER-transactivation. These data clearly showed the essential role of Med1 in mediating tamoxifen response in luminal B cells. Conclusions: These data demonstrate a novel role of Med1 in promotion of tamoxifen-induced growth of luminal B breast cancer cells and suggest that inhibiting Med1/or its activation may prove to be beneficial to overcome/reduce poor prognosis of basal B breast tumors. This conclusion is supported by in vivo data showing overexpression of Med1 in luminal B estrogen receptor-positive breast tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-57.

Abstract #3044 Introduction: Obesity is an independent risk factor for breast cancer and obese breast cancer patients exhibit a higher risk for larger tumor burden, increased metastasis and poor response to endocrine therapy. Obesity affects breast carcinogenesis by autocrine and paracrine effects of adipocytokine leptin and IGF-1. We have previously shown that leptin induces growth stimulation of breast cancer cells by recruiting histone acetyltransferases and mediator complex to cyclin D1 promoter via activation of Stat3. In the present study, we found a novel bidirectional crosstalk between IGF-1 and leptin signaling that promotes invasion and migration of triple-negative (ER, PR, HER2 negative) breast cancer cells; MDA-MB-231, MDA-MB-468, MDA-MB-435 and HCC-1806. Methods and Results: IGF-1 induced significant tyrosine phosphorylation of leptin receptor (Ob-Rb) and leptin induced tyrosine phosphorylation of IGF-1 Receptor (IGF-1R). Combined treatment of leptin and IGF-1 induced synergistic activation of both Ob-Rb and IGF-1R along with activation of Akt and ERK. Furthermore, IGF-1 and leptin synergistically transactivated epidermal growth factor receptor (EGFR) and induced proliferation of triple negative breast cancer cells. We also found that tyrosine phosphorylation of EGFR induced by combined treatment of leptin and IGF-1 was significantly inhibited not only by EGFR tyrosine kinase inhibitor, AG1478 but also with a broad-spectrum matrix metalloproteinase inhibitor, GM6001 indicating that leptin and IGF-1 induced EGFR transactivation is dependent on proteolytic release of EGFR ligands. Intriguingly, we also found that combined treatment of leptin and IGF-1 potently induced invasion of triple negative breast cancer cells in Matrigel invasion and quantitative Electric Cell-Substrate Impedance Sensing invasion assays. Inhibition of leptin and IGF-1 mediated EGFR activation using a highly potent, reversible inhibitor of HER1/EGFR tyrosine kinase, erlotinib or dual epidermal growth factor receptor (EGFR)/human EGFR-2 (HER2) kinase inhibitor, lapatinib effectively inhibited leptin and IGF-1 induced invasion. Also, inhibition of EGFR activation using erlotinib and lapatinib reduced leptin- and IGF-1-induced migration of triple negative breast cancer cells. Discussion: Taken together these data indicate a novel bidirectional crosstalk between leptin and IGF-1 signaling that transactivates EGFR and promotes metastatic properties, invasion and migration of triple negative breast carcinoma cells. Our novel findings indicate the possibility of using EGFR inhibitors erlotinib and lapatinib to counter the pro-cancerous effects of leptin and IGF-1 signaling in triple negative breast cancers. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3044.

Abstract #3067 Introduction: The prevalence of obesity in the developed world has reached epidemic proportions in recent years. Obese breast cancer patients exhibit a higher risk for metastasis, larger tumor burden and poorer response to endocrine therapy. Therefore, it is very important to understand the adverse effects of obesity on breast cancer in order to devise appropriate new approaches to their treatment. Obesity is considered an endocrine disorder and serum levels of adiponectin, an adipocytokine proposed to have therapeutic potential, get significantly lowered with obesity. More recently, low serum adiponectin levels were associated with increased risk and more aggressive phenotype (larger tumor size and high histological grade) of breast cancer in women. Recently, anti-proliferative and pro-apoptotic response of adiponectin was shown in breast cancer cells but the molecular mechanisms governing the anti-cancer effects of adiponectin largely remain unknown. In the present study, we found a novel mechanism underlying adiponectin action involving upregulation of tumor suppressor LKB1 and activation of AMPK (AMP-activated protein kinase) signaling to inhibit malignant properties of breast cancer cells. Methods and Results: Adiponectin inhibited malignant properties such as invasion and migration of breast cancer cells in matrigel invasion, scratch migration and quantitative Electric Cell-Substrate Impedance Sensing (ECIS) invasion and migration assays. Interestingly, adiponectin induced phosphorylation of AMPK at Thr 172 in breast cancer cells. Adiponectin treatment also affected downstream targets of mTOR signaling pathway, including decreased phosphorylation of p70S6K showing the involvement of mTOR-S6K axis. Intriguingly, we found that adiponectin increased the expression of tumor suppressor LKB1 in breast cancer cells. Co-immunoprecipitation assays revealed that LKB1 co-immunoprecipitated with tumor suppressor TSC2. Increase in expression of upstream kinase LKB1 might directly regulate adiponectin induced activation of AMPK. We also found that breast cancer cells transfected with wild-type LKB1 plasmids increased phosphorylation of AMPK and decreased phosphorylation of p70S6K showing decrease in mTOR activity. In contrast, breast cancer cells transfected with kinase dead LKB1 decreased phosphorylation of AMPK and increased activation of p70S6K. Discussion: Taken together these data indicate a novel crosstalk between adiponectin and tumor suppressor LKB1 that modulates AMPK-S6K axis and inhibits metastatic properties, invasion and migration of breast cancer cells. The influence of adipocytokine, adiponectin on breast carcinogenesis is just beginning to be elucidated and our study is the first to show the direct involvement of tumor suppressor genes LKB1 and TSC2 in anti-cancer effects of adiponectin. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3067.

Introduction: Obesity is an independent risk factor for breast cancer and obese breast cancer patients exhibit a higher risk for larger tumor burden, increased metastasis, and poor response to endocrine therapy. Obesity affects breast carcinogenesis by autocrine and paracrine effects of adipocytokine leptin and IGF-1. We have previously shown that leptin induces growth stimulation of breast cancer cells by recruiting histone acetyltransferases and mediator complex to cyclin D1 promoter via activation of Stat3. In the present study, we found a novel bidirectional crosstalk between IGF-1 and leptin signaling that promotes invasion and migration of triple-negative (ER, PR, HER2 negative) breast cancer cells MDA-MB-231, MDA-MB-468, MDA-MB-435, and HCC-1806. Methods and Results: IGF-1 induced significant tyrosine phosphorylation of leptin receptor (Ob-Rb) and leptin induced tyrosine phosphorylation of IGF-1 receptor (IGF-1R). Combined treatment of leptin and IGF-1 induced synergistic activation of both Ob-Rb and IGF-1R along with activation of Akt and ERK. Furthermore, IGF-1 and leptin synergistically transactivated epidermal growth factor receptor (EGFR) and induced proliferation of triple negative breast cancer cells. We also found that tyrosine phosphorylation of EGFR induced by combined treatment of leptin and IGF-1 was significantly inhibited not only by EGFR tyrosine kinase inhibitor AG1478 but also with a broad-spectrum matrix metalloproteinase inhibitor, GM6001, indicating that leptin and IGF-1 induced EGFR transactivation is dependent on proteolytic release of EGFR ligands. Intriguingly, we also found that combined treatment of leptin and IGF-1 potently induced invasion of triple negative breast cancer cells in Matrigel invasion and quantitative Electric Cell-Substrate Impedance Sensing invasion assays. Inhibition of leptin and IGF-1 mediated EGFR activation using a highly potent, reversible inhibitor of HER1/EGFR tyrosine kinase (erlotinib) or dual EGFR/HER2 kinase inhibitor (lapatinib) effectively inhibited leptin and IGF-1 induced invasion. Also, inhibition of EGFR activation using erlotinib and lapatinib reduced leptin- and IGF-1 induced migration of triple negative breast cancer cells. Conclusions: Taken together, these data indicate a novel bidirectional crosstalk between leptin and IGF-1 signaling that transactivates EGFR and promotes metastatic properties, invasion, and migration of triple negative breast carcinoma cells. Our novel findings indicate the possibility of using EGFR inhibitors erlotinib and lapatinib to counter the pro-cancerous effects of leptin and IGF-1 signaling in triple negative breast cancers.